Most mitochondrial proteins are made outside the organelle and must be imported through the mitochondrial double membrane and there assembled into active enzyme complexes. This project studies the synthesis of mitochondrial inner membrane enzyme complexes, particularly the proton translocating ATPase, on two levels: 1) assembly of the complex from imported subunits and subunits made inside the organelle; 2) nuclear gene expression required for the synthesis and transport of mitochondrial proteins made in the cytosol. Specifically the project entails: (A) Reproducing the assembly of the mitochondrial ATPase complex in vitro by adding higher molecular weight precursors of the cytoplasmically made subunits to isolated mitochondria. (B) Isolation and chracterization of mutants in which the import and processing of externally made mitochondrial proteins is impared. (C) Analysis of mutants in which respiration is uncoupled from oxidative phosphorylation. (D) Isolation of genes encoding ATPase subunits using recombinant DNA techniques. This study employs the yeast Saccharomyces cerevisiae because it permits selection of mutants with altered mitochondrial function, and because of the facility of this organism for genetic and biochemical manipulation. This system provides one of the few opportunities to study the genetic regulation of membrane biosynthesis in eukaryotes. Ultimately, we hope to achieve a high resolution of this process in eukaryotes as is available in E. coli. Understanding the control of membrane protein synthesis has medical relevance to pathological conditions in which cellular membranes are altered, such as viral infection and neoplasia. In addition, this project is applicable to physiologic states, such as cardiachypertrophy, in which the respiratory capacity of cells is perturbed.